The long-term objective of this project is to understand how extracellular stimuli can induce a single gene to encode functionally distinct protein isoforms through the process of signaling induced alternative splicing. Alternative splicing is a major determinant of diversity within the human proteome and is a critical mechanism for modulating protein expression. In particular, an increasing number of genes have been shown to undergo alternative splicing in response to extracellular stimuli. Moreover, mistakes in alternative splicing have been linked to numerous human diseases. Despite the significance of alternative splicing to human health, relatively little is understood about the mechanisms that regulate this process. This proposal seeks to understand the regulated splicing of the CD45 gene that occurs in response to T cell activation, as a model for signaling-induced regulation of alternative splicing. The Specific Aims of this proposal are 1) To identify the cis-regulatory sequences that determine the pattern of CD45 splicing, 2) To identify and characterize the RNA-binding proteins that regulate CD45 splicing, and 3) To identify components of the pathway(s) by which T cell activation induces changes in CD45 splicing. The sequence requirements for CD45 regulation will be determined by assaying the effects of mutations within CD45 minigenes on the splicing of CD45. Protein that bind to the functional elements within CD45 RNA will be identified by biochemical purification, and both in vitro and cell-based functional assays will be used to determine the mechanisms by which these proteins regulate CD45 splicing. Finally, a cell-based screen will be used to identify and characterize mutant cells that are deficient in activation-induced alternative splicing, in order to determine the pathway(s) that lead from the initial signaling event to the regulation of splicing. Together these studies will lead to greater insight as to the mechanisms by which activation of signaling pathways leads to regulation of alternative splicing, and in so doing, are likely to suggest potential targets for therapeutic modification of pre-mRNA splicing.